Obesity can lead to several cardiovascular problems including hypertension and left ventricular hypertrophy. In addition, increased heart rate and plasma catecholamines, and reduced heart rate variability in obesity are due to altered cardiac autonomic control including increased sympathetic and reduced parasympathetic, activity. Abnormal autonomic drive is a causal factor in hypertension and congestive heart failure, and may similarly promote cardiovascular disturbances in obesity. Neuropeptides such as neuropeptide Y (NPY) and galanin, released from sympathetic terminals in the heart, can inhibit acetylcholine release from adjacent parasympathetic nerves. Increased sympathetic drive in obesity may therefore augment neuropeptide release from atrial nerves, thereby promoting the inhibition of acetylcholine release from parasympathetic terminals. This study examines the role of sympathetic neuropeptides in inhibiting both short-term release, and long-term synthesis, of the parasympathetic neurotransmitter acetylcholine in the diet-induced obesity-prone rat heart. An atrial explant system will be utilized to stimulate release of neurotransmitters and neuropeptides from atrial terminals and these proteins will be measured with a sensitive HPLC-MS method. The development of a non-radioactivity based, sensitive assay for simultaneous measurements of absolute levels of neurotransmitters and neuropeptides, is an important novel approach that will be established through this study. We will also co-stimulate autonomic peripheral outflow in vivo to examine functional consequences of parasympathetic disturbances in obesity. Laser capture microdissection of parasympathetic cardiac ganglion neurons followed by qRT-PCR will allow examination of cholinergic marker genes in obesity. Cultured cardiac ganglion neurons will have neuropeptide receptor genes knocked down to examine neuropeptide regulation of acetylcholine synthesis and transport. Attenuated parasympathetic function can result in dysregulated heart rate control, potential for arrhythmias and indirectly contribute to myocardial dysfunction from catecholamine toxicity and increased workload. These studies should identify novel neuropeptide targets for reversing abnormal parasympathetic activity in obese subjects. In addition, a sensitive HPLC-MS method for simultaneous determination of neurochemicals will be developed.